Maximum likelihood phylogenetic trees for mammalian <i>Cδ</i> exons.

<p>A) <i>δCH2</i>, B) <i>δCH3</i>, C) <i>Cδ</i> secretory exon, D) <i>Cδ</i> transmembrane exon 1. Platypus included as an outgroup in D. Bootstrap values from 1000 replicates appear next to the nodes.</p

Rabbit remnant <i>Cδ</i> transmembrane (<i>δTM1</i> and <i>δTM2</i>) exons.

<p>A) Nucleotide sequence alignment of rabbit remnant <i>δTM1</i> exon with <i>δTM1</i> exons of various mammalian species. Potential 5’ and 3’ splice sites in rabbit <i>δTM1</i> enclosed in boxes. B) Amino acid sequence alignment of rabbit and other mammalian <i>δTM1</i> exons. RF1 and RF2 indicate reading frames 1 and 2 for rabbit <i>δTM1</i>. Asterisks indicate stop codons. C) Nucleotide sequence alignment of rabbit remnant <i>δTM2</i> exon with <i>δTM2</i> exons of various mammalian species. Potential 5’ splice site in rabbit <i>δTM2</i> enclosed in box. Translated region in A) and C) indicated by uppercase letters. dashes, nucleotide or amino acid gaps.</p

The remnant of the European rabbit (<i>Oryctolagus cuniculus</i>) IgD gene

<div><p>Although IgD first appeared, along with IgM, in the cartilaginous fishes and has been retained throughout subsequent vertebrate evolution, it has been lost in a diverse group of vertebrate species. We previously showed that, unlike vertebrates that express IgD, the rabbit lacks an IgD (<i>Cδ</i>) gene within 13.5 kb downstream of the IgM gene. We report here that, by conducting BLAST searches of rabbit Ig heavy chain genomic DNA with known mammalian IgD exons, we identified the remnant of the rabbit <i>Cδ</i> gene approximately 21 kb downstream of the IgM gene. The remnant <i>Cδ</i> locus lacks the <i>δCH1</i> and hinge exons, but contains truncated <i>δCH2</i> and <i>δCH</i>3 exons, as well as largely intact, but non-functional, secretory and transmembrane exons. In addition, we report that the <i>Cδ</i> gene probably became non-functional in leporids at least prior to the divergence of rabbits and hares ~12 million years ago.</p></div

Nucleotide positions of rabbit remnant <i>Cδ</i> exons and neighboring DNA elements in BAC clone 27N5.

<p>Nucleotide positions of rabbit remnant <i>Cδ</i> exons and neighboring DNA elements in BAC clone 27N5.</p

The remnant of the rabbit IgD locus.

<p>The remnant of the rabbit <i>Cδ</i> gene and the inserted repetitive DNA elements downstream of the <i>Cμ</i> transmembrane exons are depicted. The rabbit <i>Cδ</i> exons are represented by black rectangles. White rectangles adjacent to the <i>δCH2</i> and <i>δCH3</i> exons indicate regions of loss of similarity to their homologs in other mammalian species (the non-homologous region of <i>δCH2</i> overlaps L1MA9 5). Repetitive DNA elements are labeled and designated by colored rectangles. The directionality of C repeats, and direct repeats flanking the 5’-most endogenous retrovirus, are indicated by arrowheads. The putative σδ switch region is indicated by a hatched rectangle. <i>δS</i>, <i>Cδ</i> secretory exon; <i>δTM1</i>, <i>Cδ</i> transmembrane exon 1; <i>δTM2</i>, <i>Cδ</i> transmembrane exon 2; <i>μTM1</i>, <i>Cμ</i> transmembrane exon 1; <i>μTM2</i>, <i>Cμ</i> transmembrane exon 2; ψHMG14, ψASB, ψMAN, high-mobility group 14, arylsulfatase B and endo-alpha-like mannosidase processed pseudogenes, respectively; σδ, putative σδ switch region; nomenclature for C repeats (and the LINE1, L1OcμM) follows that adopted in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182029#pone.0182029.ref007" target="_blank">7</a>] (C13 was not present in the genomic DNA used in that study); nomenclature of other repetitive DNA elements follows that used by the Genetic Information Research Institute [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182029#pone.0182029.ref032" target="_blank">32</a>].</p

RT-PCR and 3’RACE analysis of the rabbit remnant <i>δCH2</i>, <i>δCH3</i>, <i>Cδ</i> secretory (<i>δS</i>) and <i>Cδ</i> transmembrane 1 (<i>δTM1</i>) exons.

<p>A) RT-PCR analysis of rabbit <i>δCH2</i> and <i>δCH3</i>. RT-PCR was performed using a forward primer in <i>V</i>_<i>H</i><i>1</i> (the preferentially rearranged rabbit <i>V</i>_<i>H</i> gene segment) and reverse primers in <i>Cμ</i> (lane 2; positive control), <i>δCH2</i> (lane 3), or <i>δCH3</i> (lane 4). Lane 5 (neg), negative control (same primers as lane 2, with no template). cDNA prepared from appendix of a 6-week-old rabbit. Forward primers: VDJ-Cμ Nest f2; reverse primers: VDJ-Cμ Nest r2, RbDelta CH2 r2, RbDelta CH3 r; Marker band sizes indicated in base pairs (bp). B) 3’RACE analysis of <i>δS</i> and <i>δTM1</i>. 3’RACE was performed for <i>δS</i> (lanes 1–3), <i>δTM1</i> (lanes 4–6) and <i>Cμ</i> (lanes 7–9; positive control) using cDNA prepared from appendix (Apx), spleen (Spl) and bone marrow (BM) of a 6-week-old rabbit. Forward primers: RbDelta SecExon 3’RACE f1, RbDelta SecExon 3’RACE f2, RbDelta TM1 3’RACE f1, RbDelta TM1 3’RACE f2, Cmu f, Mid mu f; Lane 10 (neg), negative control (same primer pair as lanes 7–9, with no template). Marker band sizes indicated in base pairs (bp). 3’RACE primers and protocol as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182029#pone.0182029.ref033" target="_blank">33</a>].</p

Alignment of rabbit and hare remnant <i>δCH2</i> exons with the <i>δCH2</i> exons of various mammalian species.

<p>Nucleotide positions are numbered above each panel. The region with no similarity to other mammalian <i>δCH2</i> exons is highlighted in gray. The region of overlap with L1MA9 5 enclosed in box.</p